Deciphering the pre- leukaemic transcriptional framework shaped by the cebpa n321d mutation

Advances in genomic profiling have provided comprehensive lists of genetic aberrations associated with Acute Myeloid Leukaemia (AML). Nevertheless, relating the genetic changes to the regulatory networks that favour leukemic transformation remains a challenge. For example, mutations in transcription factor (TF) C/EBPα are observed in 10% of patients with AML and are believed to be an early pre-leukemic event. However, little is known about the early steps of mutant C/EBPα induced leukemogenesis. We aim to gain insights into this question by utilizing a previously defined in vitro model of multipotent progenitors (HOXB8-FL) and focusing on the C/EBPα N321D mutation that was shown to lead to an aggressive AML in mouse models (Togami et al., 2015). Using differentiation assays of parental HOXB8-FL and HOXB8-FL transduced with the mutant N321D we show that the CEBPA N321D-transduced cells demonstrate a growth advantage in differentiation medium. Flow cytometry and single-cell transcriptomic characterisation of cells at different time points revealed that the mutation stalls the multipotent HOXB8-FL cell line along the differentiation trajectory resulting in a self-renewing progenitor-like state exhibiting haematopoietic markers indicative of a plasmacytoid dendritic cell-like phenotype. We also demonstrate that CEBPA N321D exhibits leukaemogenic potential in vivo as mouse recipients injected with CEBPA N321D cells develop clinical signs of leukaemia, including high white-blood count, splenomegaly and hepatomegaly. To understand how CEBPA N321D reshapes the molecular architecture we are in the process of performing a large scale CRISPR/Cas9 screen with 79 TFs. Analysis of transcriptional signatures of the single knock-out of TFs should allow us to build a comprehensive TF map to better understand the regulatory network that drives and maintains the pre-leukemic state. Advances in genomic profiling have provided comprehensive lists of genetic aberrations associated with Acute Myeloid Leukaemia (AML). Nevertheless, relating the genetic changes to the regulatory networks that favour leukemic transformation remains a challenge. For example, mutations in transcription factor (TF) C/EBPα are observed in 10% of patients with AML and are believed to be an early pre-leukemic event. However, little is known about the early steps of mutant C/EBPα induced leukemogenesis. We aim to gain insights into this question by utilizing a previously defined in vitro model of multipotent progenitors (HOXB8-FL) and focusing on the C/EBPα N321D mutation that was shown to lead to an aggressive AML in mouse models (Togami et al., 2015). Using differentiation assays of parental HOXB8-FL and HOXB8-FL transduced with the mutant N321D we show that the CEBPA N321D-transduced cells demonstrate a growth advantage in differentiation medium. Flow cytometry and single-cell transcriptomic characterisation of cells at different time points revealed that the mutation stalls the multipotent HOXB8-FL cell line along the differentiation trajectory resulting in a self-renewing progenitor-like state exhibiting haematopoietic markers indicative of a plasmacytoid dendritic cell-like phenotype. We also demonstrate that CEBPA N321D exhibits leukaemogenic potential in vivo as mouse recipients injected with CEBPA N321D cells develop clinical signs of leukaemia, including high white-blood count, splenomegaly and hepatomegaly. To understand how CEBPA N321D reshapes the molecular architecture we are in the process of performing a large scale CRISPR/Cas9 screen with 79 TFs. Analysis of transcriptional signatures of the single knock-out of TFs should allow us to build a comprehensive TF map to better understand the regulatory network that drives and maintains the pre-leukemic state.